Tyrosine specific protein kinases are encoded by certain retrovirus transforming genes, such as the src gene of Rous sarcoma virus. These proteins are believed to transform by phosphorylating specific cellular components. The overall objective of this project is to determine if protein phosphorylation at tyrosine is necessary for transformation and to identify those cellular targets which have a significant role in transformation. Three different approaches will be used. The initial part of this work is concerned with the characterization of the proteins phosphorylated at tyrosine in cells transformed by different retroviruses or infected by retrovirus vectors carrying the cellular homologs of these genes. The objectives of these initial studies are to determine whether changes in substrate specificity are associated with the ability to transform, whether transforming proteins with partial homology to the RSV src gene also have associated kinase activity, and whether phosphorylation at tyrosine modifies the function of the phosphorylated polypeptide. Second, protein phosphorylation at serine in retrovirus transformed cells will be characterized in order to examine the hypothesis that serine phosphorylation represents a secondary event which mediates some of the actions of viral tyrosine kinases. The activity of the Ca++- and phospholipid-dependent protein kinase C in retrovirus-transformed cells will be examined in order to determine if this enzyme is involved in transformation. Third, cellular mutants with altered susceptibility to retrovirus transforming proteins will be isolated in order to define cellular genes whose products interact directly with retrovirus transforming proteins. Transformed cells will be selected from cell lines infected by RSV mutants that carry partial or conditional defects in the src gene. If cellular mutants in which the viral defect is suppressed by a cellular mutation can be identified, DNA transfer experiments will be carried out to determine if isolation of these genes by molecular cloning is feasible.